World J Microbiol Biotechnol DOI 10.1007/s11274-013-1413-2 SHORT COMMUNICATION Growth, antioxidant capacity and total carotene of Dunaliella salina DCCBC15 in a low cost enriched natural seawater medium Duc Tran • Nguyen Doan • Clifford Louime Mario Giordano • Sixto Portilla • Received: 28 February 2013 / Accepted: 21 June 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Dunaliella is currently drawing worldwide attention as an alternative source of nutraceuticals Commercially, b-carotene making up over 10 % of Dunaliella biomass is generating the most interest These compounds, because of their non-toxic properties, have found applications in the food, drug and cosmetic industry The b-carotene content of Dunaliella cells, however, depends heavily on the growth conditions and especially on the availability of nutrients, salinity, irradiance and temperature in the growth medium A chemically well defined medium is usually required, which significantly contributes to the cost of pigment production; hence a desire for low cost marine media The present study aimed at evaluating the suitability of six different media, especially exploiting local potential resources, for the mass production of Dunaliella salina DCCBC15 as functional food and medicine The efficacy of a new selected low-cost enriched natural seawater medium (MD4), supplemented with industrial N–P–K fertilizer, was investigated with respect to D Tran (&) Á N Doan School of Biotechnology, International University, VNU, Thu Duc Dist., Ho Chi Minh, Vietnam e-mail: tnducminh@yahoo.com; tnduc@hcmiu.edu.vn C Louime FAMU BioEnergy Group, College of Engineering Sciences, Technology and Agriculture, Florida A&M University, Tallahassee, FL 32307, USA M Giordano Dipartimento di Scienze della Vita e dell’Ambiente (DISVA), Universita` Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy S Portilla Center for Estuarine, Environmental and Coastal Oceans Monitoring, Dowling College, 150 Idle Hour Blvd., Oakdale, NY 11769, USA biomass production, chlorophyll, antioxidant capacity, and total carotene by Dunaliella though culture conditions were not optimized yet This new medium (MD4) appears extremely promising, since it affords a higher production of Dunaliella biomass and pigments compared with the control, a common artificial medium (MD1), while allowing a substantial reduction in the production costs The medium is also recommended for culturing other marine algae Keywords Dunaliella Á Carotene Á Medium Á Cultivation Introduction Dunaliella is a cell wall-less green flagellate belonging to the order Volvocales (Chlorophyceae, Chlorophyta) (Oren 2005; Polle et al 2009) There are about 26 saltwater species described for the genus of Dunaliella (Oren 2005; Borowitzka and Siva 2007; Polle et al 2009) D salina TEODORESCO is a model organism and the type species of the genus, whose vegetative cells are under high irradiance, high salinity, or low nitrogen concentrations, are characterized by an intense orange color due to the intracellular accumulation of large amounts of b-carotene (Polle et al 2009; Lamers et al 2010; Fu et al 2013) D salina are among the main sources for natural b-carotene, which is employed in the food, cosmetic and pharmaceutical industries and also used as colorant, antioxidant and anti-cancer agent (Ben-Amotz et al 1982, 1989; Borowitzka and Borowitzka 1988; Sergio and Rubens 2010; Fu et al 2013) Dunaliella history, biological characteristics as well as applications were presented in thorough reviews and publications of authors such as Oren (2005); Polle et al (2009), and others Various artificial and natural seawater media have been used for Dunaliella cultivation, both in laboratory settings 123 World J Microbiol Biotechnol and in large industrial scales (Ben-Amotz 1995; Pisal and Lele 2005; Garcı´a-Gonza´lez et al 2003; Simental and Sa´nchez-Saavedra 2003; Fazeli et al 2006; Grobbelaar 1995; Abu-Rezq et al 2010; Ana Prieto et al 2011) However, in developing countries such as Vietnam, some of these media are unrealistically expensive and the quest to exploit local, lower cost medium components may be crucial for a commercially viable cultivation of Dunaliella Vietnam has a vast potential for Dunaliella b-carotene production along its coastal areas This would contribute to the economic and environmental stability of the country (and beyond) In the present study, we investigated the growth of Dunaliella using local seawater enriched with an industrial fertilizer (N–P–K) commercially available in Vietnam In addition to N, P and K, this fertilizer contains micronutrients (Mg, Ca, Zn, Cu, Fe, Mo) present in artificial medium and essential for Dunaliella growth (Borowitzka and Borowitzka 1988; Sergio and Rubens 2010) The growth data showed that our medium allows to attain a much lower cost per unit of dry mass than more common artificial media The medium is recommended for the culturing of other marine algae as well batch and maintained in artificial 1.5 M NaCl medium according to Chitlaru and Pick (1989) Briefly, the medium contained 1.5 M NaCl; 0.4 M Tris–HCl, mM KNO3, mM MgSO4, 0.3 mM CaCl2, 0.2 mM KH2PO4, 1.5 lM FeCl3 in lM EDTA, 0.185 mM H3BO3, lM MnCl2, 0.8 lM ZnCl2, 0.2nM CuCl2, 0.2 lM Na2MoO4, 20nM CoCl2, 50 mM NaHCO3 Six different 1.5 M NaCl media were devised for D salina cultures, including one fully artificial medium (MD1), and five media based on natural seawater, enriched by the addition of different chemicals (MD2–MD6), the detailed composition of all media is shown in Table Components of N–P–K are listed as a note below Table MD6 is enriched natural seawater with components according to Garcı´a-Gonza´lez et al (2003) and Ana Prieto et al (2011) for open pilot culture The N–P–K (code: Dau Trau 501) was bought from Binh Dien fertilizer company Seawater with salinity about 0.5 M collected from Long Hai beach, Vung Tau province, was first filtered to remove sand and other small particles Then all media (added salts to obtain 1.5 M) were sterile filtered using cellulose acetate filter with pore size 0.45 lm after adding chemicals according to Table Materials and methods Experimental design Dunaliella salina DCCBC15 growth conditions One liter of algal culture was grown in liter flasks; for each treatment, replicate cultures were used The cultures were maintained at 25 °C, and at a photon flux density of 50 lmol m-2 s-1 (PAR), provided by white fluorescence lamps The experiment was run for 24 days Dunaliella salina DCCBC15 was kindly provided by Dr E.W Polle, Department of Biology, Brooklyn College of CUNY Brooklyn, NY (USA) The cultures were grown in Table Components of the different media tested in this study Chemicals Medium MD1 Artificiala (Tris–HCl) MD2 Natural SW added with artificial components as in MD1 (Tris–HCl) MD3 Natural SW added with artificial components as in MD1 (but no Tris–HCl) MD Natural SW (no Tris– HCl) (g/l) MD5 Natural SW (no Tris– HCl) (g/l) MD6 Natural SW (no Tris– HCl) (g/l) N–P–Kb – – – 0.1 – – MgS04 – – – 1.86 1.86 – EDTA – – – 0.00876 0.00876 – FeCl3 – – – 0.00049 0.00049 – MnCl2 – – – 0.00189 0.00189 – NaHC03 NaN03 – – – – – – 3.15 – 3.15 – 0.084 0.1275 NaH2P04 – – – – – 0.012 FeCl3Á6H20 – – – – – 0.00324 a 87.75 g/l NaCl; 9.48 g/l Tris–HCl, 0.6675 g/l KNO3, 1.86 g/l MgSO4, 0.06615 g/l CaCl2, 0.022 g/l KH2PO4, 9.75 10-4 g/l FeCl3 in 8.76 10-3 g/l EDTA, 6.975 10-4 g/l H3BO3, 9.45 10-5 g/l MnCl2, 8.16 10-6 g/l ZnCl2, 3.834 10-6 g/l CuCl2, 3.6 10-5 g/l Na2MoO4, 3.567 10-6 g/l CoCl2, 3.15 g/l NaHCO3 b N–P–K (30–15–10): 30 % N, 15 % P2O5, 10 % K2O, 0.05 % Mg, 0.05 % Ca, 0.01 % B, 0.05 % Zn, 0.05 % Cu, 0.05 % Fe, 0.025 % Mn, 0.005 % Mo, a-NAA, b-NOA, GA3 123 World J Microbiol Biotechnol Table Concentration (mg/l) of some components in N–P–K compared with artificial medium (MD1) Elements N–P–K (mg/l) Artificial medium (mg/l) N 30 70 P 6.55 3.2 K 8.3 195 percent according to the formula I % = (Ablank - Asample)/ Ablank 100 Growth and biomass determination Mg 0.05 120 Ca 0.05 12 Zn 0.05 0.052 Cu Fe 0.05 0.05 0.0192 0.112 Mn 0.025 0.55 Mo 0.005 1.35 a-NAA 0.1 N/A b-NOA 0.1 N/A GA3 0.1 N/A Cell counts were performed every days using haemacytometer For biomass determination, the cultures were filtered through 47 mm glass fiber filters with a 0.7 lm nominal pore size The filter was washed with ml of ammonium formiate (0.5 M), dried at 103 °C for h or until weight was stable, for dry weight determination The dry biomass was further burned in a furnace at 550 °C to determine the ash weight The biomass was calculated as the difference of the dry weight and the ash weight Statistical analysis Pigments analysis Pigment extraction was carried out according to Psal and Lele (2005) A ml aliquot of Dunaliella salina culture was centrifuged at 8,000 rpm for 20 The pellet was washed with distilled water, suspended in an 80 % acetone solution, thoroughly vortexed and centrifuged to extract pigments until the pellets turned clear/white The absorbance of the relevant pigments was measured in the extract using UV–VIS spectrophotometer Chlorophyll and total carotene were estimated according to Lichtentaler and Wellburn formulas (1985): Data were analyzed by one-way ANOVA using the SPSS software The level of significance was always set at P \ 0.05 Results Growth where Chla chlorophyll a, Chlb chlorophyll b, Cx ? c total carotene Results of the different media with respect to cell density, biomass and chlorophyll a after 24 days of cultivation, which were all in line, are shown in Fig The medium (MD4) enriched with industrial fertilizer N–P–K showed best overall performance The differences of cell density, biomass and chlorophyll a among the different media tested were statistically significant (P = 0.021 \ 0.05) The highest cell numbers, biomass and chlorophyll a in MD5, MD6 were 0.5 106/ml, 0.2 g/l and lg/ml respectively, which were three to four times lower than those in MD1, MD4 after weeks (Fig 1a, b) Antioxidant capacity Antioxidant capacity and total carotene DPPH solution was prepared by dissolving 0.004 g of DPPH in 100 ml of methanol Five ml of D salina centrifuged The pellet was extracted with 20 ml of absolute ethanol and vortexed well The extract was filtered to remove possible particles would interfere optical density reading, and dried at a temperature of 55 °C The dried extract was dissolved in ml of absolute ethanol The absorbance of the extract at 515 nm was determined spectrophotometrically A blank sample (absolute ethanol) was also taken as control The antioxidant activity was calculated based on the inhibition of free radical DPPH in Similar to growth, antioxidant capacity and carotene of Dunaliella were significantly lower in natural media MD5, MD6 (P = 0.00) These media therefore not seem to be appropriate for Dunaliella cultivation For other media MD1, MD2, MD3, MD4, though growth of Dunaliella declined after the first initial weeks due to limiting nutrients (Fig 1), antioxidant capacity and carotene continued to accumulate for an additional week due to limiting nutrients (Fig 2b) Among these four media, antioxidant capacity and carotene of Dunaliella were higher in artificial MD1 and natural MD4 after the exponential growth phase Chla lg=mlị ẳ 11:75 A662 ị 2:35 A645 ị Chlb lg=mlị ẳ 18:61 A645 ị 3:96 A662 ị Cx ỵ c lg=mlị ẳ 1000A470 2:270 Chla À 81:4 ChlbÞ=198 123 World J Microbiol Biotechnol Fig Cell density (a), biomass (b) and chlorophyll a (c) of Dunaliella bardawil grown in six different media (MD1–MD6) Fig Antioxidant activity (a) and total carotene (b) of D salina grown in six different media (Fig 2) Antioxidant capacity and carotene yield in MD1, MD4 were almost twice higher than those in MD5, MD6, which were 50 % and [4 lg/ml respectively Discussion Low cost medium for various algae based on fertilizers have been investigated and shown to be potential for lab and large scale cultivation of various groups of algae (Garcı´a-Gonza´lez et al 2003; Simental and Sa´nchez-Saavedra 2003) However using direct local resources regarding low cost chemicals and natural seawater should be more practical and at least one step closer for industrial development With the abundance of natural seawater in Vietnam, it provides a great opportunity to develop large 123 scale cultivation of Dunaliella (and other algae) for various application such as feed in aquaculture, food, medical uses, and even biofuel (Takagi et al 2006; Chen et al 2011), which all together promoted us to initiate this study From all media devised in this study, Dunaliella adapted and started to grow after days However, it performed best in the MD4 medium of seawater enriched with N–P–K fertilizer This was revealed through biomass, cell density and chlorophyll a data significantly at P \ 0.05 (Fig 1), as all cultures reached stationary phase after weeks and started collapsing/dead phase which are common due to nutrient limit or inhibitory metabolites releasing from algal culture (Brown et al 1993; Sergio and Rubens 2010; Chen et al 2011; Tran et al unpublished data) Growth of Dunaliella in MD6 medium [which was used in open cultivation by Garcı´a-Gonza´lez et al (2003)], and in MD5 World J Microbiol Biotechnol Table The cost of chemicals using in artificial medium Chemicals Gram/1 l medium NaCl Cost VND/1 l medium 87.75 4,914 NaHCO3 3.15 283.5 Tris–HCl 9.48 37,920 KNO3 0.6675 93.45 MgSO4 1.86 260.4 CaCl2 0.06615 5.9535 KH2PO4 0.0223344 2.456784 FeCl3 9.75 10-4 0.0741 EDTA 8.76 10-3 1.99728 -4 H3BO3 6.975 10 0.062775 MnCl2 ZnSO4 9.45 10-5 8.16 10-6 0.014175 9.792 10-4 CuCl2 3.834 10-6 1.1502 10-3 Na2MoO4 CoCl2 -5 3.6 10 0.09 -6 5.7072 10-3 3.567 10 Total 43,482 Bold indicates the key difference of costs between the two medium Table The cost of chemicals using in natural seawater medium Chemicals Gram/1 l medium Cost VND/1 l medium EDTA 8.76 10-3 1.99728 FeCl3 9.75 10-4 0.0741 NaHCO3 3.15 283.5 MnCl2 9.45 10-5 0.014175 MgSO4 1.86 260.4 Fertilizer N–P–K 30–15–10 0.1 15 NaCl Total 58.5 3,276 3,837 Bold indicates the key difference of costs between the two medium medium (which was similar to MD4 except there was no N–P–K), was much slower, which are not appropriately considered for Dunaliella cultivation The success of MD4 is underscored by the presence of N–P–K and the absence of Tris–HCl, a high cost component comprising over 90 % of media MD1 and MD2 The biomass in MD2 and MD3 dropped after 12 days which were earlier than MD1 and MD4; this could be probably that the algae were stressed under imbalance ratio of nutrients due to one of the nutrient components was additionally high coming from seawater and other nutrient(s) simultaneously run out Presumingly, growth of Dunaliella in these media (MD1, MD4) is similar, but with a cost lower than 90 % in MD4, this makes the productivity of MD4 90 % higher than MD1 (Tables 3, 4) Currently this new MD4 medium is being used more successfully in our laboratory for most marine strains from isolation to cultivation than other artificial media such as BG11, F/2, BBM, CM (Tran et al., unpublished data), which we recommended to use this medium for other marine algae as well The effectiveness of this new medium is probably due to non-defined minerals and micronutrients present in the natural seawater, which we predicted when devising the medium basing on the minimal amount of components of the artificial medium (MD1) In addition, higher concentration of phosphate, Cu and the presence of three hormones (lNAA, bNOA, GA3) in N–P– K which were not present in artificial MD1 medium (Table 2, in bold) may have further stimulatory effects on Dunaliella growth Though the new medium (MD4) was much successful compared with other media regarding low cost and higher productivity, it was not optimized for biomass and pigments production yet Further study on feeding Dunaliella with different nitrogen, phosphate salts to sustain and optimize continuous growth for optimal biomass, antioxidant and b-carotene production are on progress in our lab, which will be applied for pilot and large scale cultivation in climate conditions of Vietnam Acknowledgments The authors are grateful for the funding of Nafosted (Funding number: 106.16-2011.31) and the funding of HCM National University to carry out this research (Funding number: B2012-28-02/HD-DHQT-QLKH) The authors are also grateful to Dr Jeurgen Polle at Department of Biology, Brooklyn college for providing Dunaliella salina DCCBC15 for the study and his valuable advices The authors would like to thank anonymous reviewers of this manuscript for correction and improvement References Abu-Rezq T, Al-Hooti S, Jacob D (2010) Optimum culture conditions required for the locally isolated Dunaliella salina J Algal Biomass Utln 2:12–19 Ana Prieto J, Ca˜navate P, Garcı´a-Gonza´lez M (2011) Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes J Biotechnol 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(Chlorophyceae) The book: the alga Dunaliella: biodiversity, physiology, genomics and biotechnology Science Publishers, pp 1–14 Sergio SJ, Rubens MF (2010) Modeling growth of microalgae Dunaliella salina under different nutritional conditions Am J Biochem Biotechnol 6(4):279–283 Simental JA, Sa´nchez-Saavedra MP (2003) The effect of agricultural fertilizer on growth rate of benthic diatoms Aquacult Eng 27:265–272 Takagi M, Karseno, Yoshida T (2006) Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells J Biosci Bioeng 101(3):223–226 ... the abundance of natural seawater in Vietnam, it provides a great opportunity to develop large 123 scale cultivation of Dunaliella (and other algae) for various application such as feed in aquaculture,... 1), antioxidant capacity and carotene continued to accumulate for an additional week due to limiting nutrients (Fig 2b) Among these four media, antioxidant capacity and carotene of Dunaliella were... activity was calculated based on the inhibition of free radical DPPH in Similar to growth, antioxidant capacity and carotene of Dunaliella were significantly lower in natural media MD5, MD6 (P =